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Título
Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions
Autor(es)
Assunto
Biochemistry
Issue 130
TAT
Biotin-avidin
Cell-penetrating peptides
Pull-down
Intracellular interactions
Protein-protein interactions
Western blot
Fecha de publicación
2017
Citación
Jaraíz-Rodríguez, M., González-Sánchez, A., García-Vicente, L., Medina, J. M., Tabernero, A. (2017). Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions. Journal of Visualized Experiments, (130), e56457. doi:10.3791/56457
Resumo
[EN] Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system.
The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating
baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the
intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas.
The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we
use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the
target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that
the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However,
this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out
by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed
method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating
sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction
because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be
transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.
URI
ISSN
1940-087X
DOI
10.3791/56457
Versión del editor
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